Illumina Sequencing Protocol for Transcriptome Analysis

Total RNA extraction was performed using RNeasy plus mini kit (250) (Qiagen 74136), and mRNA isolation was accomplished using magnetic mRNA isolation kit (oligo(dT) beads; NEB E7490). The sequencing libraries were prepared using NEBNext ultra DNA library prep kit (NEB E7645S) and were sequenced on an Illumina NovaSeq 6000 using 150-bp paired-end reads. Adapter/quality trimming was performed with Trim Galore! v0.6.10 (Babraham Bioinformatics; https://github.com/FelixKrueger/TrimGalore). Quantification of transcript expressions was performed with Salmon v1.10.2 (Patro et al. 2017 (link)), and aggregation to the gene level was performed with the R package tximport v1.22.0 (Soneson et al. 2015 (link)).